The primary goals of this competing renewal application are to define the structural requirements for selective and potent binding to the glutamate synaptic vesicular transporter (VGLUT) and to advance our understanding of the structural and functional roles of VGLUT. The long-range goals of this study are to: (a) develop pharmacophore and structural models of VGLUT that will aid the development of selective molecules that can regulate vesicular storage, uptake and release of glutamate and, (b) define the role and contribution of VGLUTs and related vesicular transporter proteins in vesicle function. The proposed strategy will optimize VGLUT inhibition and binding selectivity using results from our current project combined with novel chemical, computational, biochemical and pharmacological approaches. A concurrent strategy will seek to obtain a more global understanding of VGLUT structure and function. Using integrative strategies, we will increase our project activity and broaden our efforts to define the structural, functional and pharmacological roles of VGLUT. For the proposed grant period, we will address the following objectives: Specific Aim 1: Design and synthesize inhibitor structures that selectively bind VGLUT. Emphasis will be placed on developing inhibitors that satisfy the elements of a dynamic pharmacophore model, for example, a cyclic platform with regio- and stereochemically defined placement of acid and lipophilic substituents. Specific Aim 2: Test the activity of compounds prepared in SA 1 as inhibitors of VGLUT. The ability of these compounds to bind EAA receptors and cellular transporters also will be determined to assess the specificity at VGLUT. Specific Aim 3: Generate a computationally-derived pharmacophore model of VGLUT utilizing the chemical, biochemical and pharmacological data generated in SA's 1 & 2, The pharmacophore model will be used to refine and iterate inhibitor design (SA1), affinity agents (SA4), protein probe structures (SA's 4 and 5), and to create protein 3D structural hypotheses consistent with the probe and cross-linking results (SA4 & 5). Specific Aim 4: Characterize the transmembrane domain regions of VGLUT and neighboring architectures that contribute to VGLUT structure and function. Using N-term VGLUT antibodies, affinity tags, tethered- and cross-linking probes, VGLUT residues and structures proximal to VGLUT will be isolated and identified by mass spectrometry.